野猪ASIP基因的突变位点及其与毛色表型的相关性研究

 本试验旨在研究影响动物被毛颜色变化的功能基因ASIP在野猪群体里的变异及其与被毛表型之间的相关性。通过直接测序法搜寻ASIP基因编码区、5′ UTR、3′ UTR和部分内含子区域突变位点。结果表明：在ASIP基因的3′ UTR区域识别了一个T→C突变位点（ASIP.c 695 T→C），而在其它区域没用发现。通过群体内多态性检测，7个毛色类型的野猪在此突变位点都是CC或CT基因型，而长白猪群全部是TT基因型。从这个结果得出具有不同毛色的野猪群体可能是由突变位点C等位基因引起，不能排除还有其它毛色功能基因参与野猪被毛的形成。研究为进一步了解野猪ASIP毛色功能基因在其群体内的遗传变异奠定了理论基础。     

Altered expression of melanocortin-1 receptor (MC1R) in a yellow-coloured wild raccoon dog (Nyctereutes procyonoides)

Background – The melanocortin 1 receptor (MC1R) gene plays a key role in determining coat colour in mammals by controlling the proportion of eumelanin and pheomelanin granules. Wild raccoon dogs have a mixed coat colour, with black to brown and grey hairs. Hypothesis/Objectives – The study was performed to identify the cause of the variant yellow coat colour in a wild raccoon dog. Animals – A wild raccoon dog that showed coat colour change to yellow and four wild‐type raccoon dogs that showed normal coat colour were included. Methods – To identify the cause of the variant yellow coat colour, we examined the sequence of the MC1R gene and its expression at the mRNA and protein levels. Results – The coding region of the MC1R gene of this raccoon dog comprised 954 bp, the same as for wild‐type raccoon dogs and domestic dogs. By comparing the gene with that in the wild‐type raccoon dog, a 2 bp deletion was detected in the 5′‐untranslated region, positioned 152 bp upstream of the start codon. However, there was no significant difference in the mRNA expression level. The yellow raccoon dog revealed a significantly decreased MC1R protein level compared with the wild‐type raccoon dogs, indicating an increase in pheomelanin synthesis. Conclusions and clinical importance – These results suggest that the variant coat colour in the yellow raccoon dog was associated with decreased MC1R function.     

Seven novel single nucleotide polymorphisms identified within river buffalo (Bubalus bubalis) lactoferrin gene

The present study aimed at identifying single-nucleotide polymorphic (SNP) sites in different coding and non-coding regions of lactoferrin gene in Indian riverine buffaloes. A total of 102 animals from six different river buffalo breeds were screened at six bubaline lactoferrin gene loci. Single-strand conformation polymorphism (SSCP) analysis revealed monomorphic patterns at three loci LtfE2, LtfE11, and LtfE14 while a total of eight distinct patterns were observed in the other three loci viz. LtfE5, LtfE10, and LtfE16 which correspond to respective exons and their flanking regions. Sequence analysis of different SSCP variants revealed the presence of two SNP sites within the coding (exon 16) region and five SNP sites in flanking non-coding regions (intron 4 and intron 9). Both SNPs within exon 16 were found to be synonymous. The SNPs and haplotypes identified in the present study could serve as potential markers for association with susceptibility/resistance to mastitis in buffaloes.     

ASIP蛋白与AgRP相关蛋白生物信息比较分析

利用生物信息学的方法研究人、绵羊和牛等18个物种Agouti信号蛋白（ASIP）和Agouti相关蛋白（AgRP）的编码区及其对应氨基酸序列的相关特征，以期探明两种蛋白的遗传分化特点。结果表明，在ASIP与AgRP均为399bp的CDS序列中，其核苷酸保守区域的位置分别在262～399bp和230～399bp处；ASIP和AgRP在各自CDS序列多态位点变异的各项指标数值及其百分率大小上均极为接近；在46条ASIP的CDS序列及32条AgRP的CDS序列中，二者的单倍型数分别为22种和23种，ASIP和AgRP在基因的进化以及不同物种的遗传分化上具有相似性；两者的氨基酸序列在98～140aa片段处保守性较高，该区域同时富含半胱氨酸（Cys）；ASIP和AgRP一级结构的理化性质表现为共性与差异并存；二者的肽链C端具有相似的三维结构，但各自也具有其结构特异性。     

Association analysis of novel polymorphisms in 2′, 5′‐oligoadenylate synthetase gene with reproductive traits in indigenous and cross‐bred cattle of Indian Origin

2′, 5′‐Oligoadenylate synthetases (OAS) are important components of an interferon‐mediated antiviral pathway. No polymorphisms in exonic regions of bovine OAS1 gene have been identified and associated with reproduction traits. The objective of the study was to detect and evaluate the effects of mutations in exonic region of bovine OAS1 gene with reproduction traits in cattle. DNA samples collected from 250 individual cows of two Indian dairy breeds (Sahiwal and Frieswal) of cattle were used in the study. The genetic variants of the OAS1 gene were identified with polymerase chain reaction–single‐strand conformation polymorphism (PCR‐SSCP) and sequence analysis using seven set of primer pairs. The PCR‐SSCP analysis revealed polymorphism in the fragments comprising of exon 2, exon 5 and first fragment of exon 6 while the fragments of exons 1, 3, 4 and second fragment of exon 6 were monomorphic in Sahiwal and Frieswal cattle. The mutations in the amplified region comprising of exon 2 were found to have significant association with age at first breeding and calving, service period, dry period and pregnancy rate. Significant associations were found between SNPs in the exon 5 and service and dry periods of the animal, whereas the genetic variants in the first fragment of the exon 6 showed significant association with age at first breeding and calving. To our knowledge, this study demonstrated for the first time that the polymorphisms in OAS1 gene were associated with reproductive traits and it can be chosen as a candidate gene for improvement of reproductive performance of cattle.     

Mutations in the melanocortin 1 receptor, β-defensin103 and agouti signaling protein genes, and their association with coat color phenotypes in Akita-inu dogs

To identify factors that control coat color in Akita-inu dogs, we sequenced all the exons of the melanocortin 1 receptor (MC1R), β-defensin103 (CBD103) and agouti signaling protein (ASIP) genes of dogs with four distinct coat colors, namely, brindle, sesame, red and white. Then we examined correlations among specific alleles and coat color. In the case of the MC1R gene, all white dogs were homozygous for a nonsense mutation, R306ter, while brindle, sesame, and red dogs had at least one R306 allele. In the case of the CBD103 gene, all brindle dogs were heterozygous for the G23del mutation (deletion of codon 23, encoding glycine), while all sesame and red dogs were homozygous for G23. In the case of the ASIP gene, all dogs, regardless of coat color, had at least one S82 H83 allele. A missense mutation in the ASIP gene, P87L, was identified for the first time in some Akita-inu dogs but was not associated with any specific coloration. Our results indicate that the 2 key mutations, R306ter in the MC1R gene and G23del in the CBD103 gene, are associated with the phenotypic discriminations among brindle, red/sesame, and white coats, while no mutation that might potentially be associated with the discrimination of a sesame coat from a red coat is present in the coding sequences of these three genes.     

Coat colour inheritance in horses

The colours of the horses have long been a subject of interest to owners and breeders of horses as well as to scientists. Though, the colour of horses has little to do with its performance, it is a primary means of identification and also the first indicator of questionable parentage. Probably the ancestral colour of the horse was a black-based pattern that provided camouflage protection against predators. Horse colours are mostly controlled by genes at 12 different loci. The three basic colours of horses are black, bay and chestnut. The genetic control of the basic colours of horses resides at two genetic loci, namely Extension (E) and Agouti (A) loci. Among the basic colours bay is dominant to black and both are epistatic to chestnut. Dilution of basic colours of horses as a result of four colour dilution genes such as cream dilution, dun, silver dapple and champagne resulted in extensive array of possible colours of horses. The most widespread and familiar of the horse colour dilution gene is the one that produces the golden body colour and are called as palomino or buckskin based on the colour of the points. The grey coat colour is due to the presence of dominant gene (G) at the grey locus. Grey is epistatic to all coat colour genes except white and a grey horse must have at least one grey parent. Roan is due to a dominant gene (Rn) at roan locus and this combines with any base colour to produce the various shades of roan pattern. White coat is due to a single dominant gene (W) and it is epistatic to the genes controlling all other colours. White marking in the face and legs are due to genetic and non-genetic factors. Several genes are involved in producing white markings. During recent years, comparative genomics and whole genome scanning have been used to develop DNA tests for different variety of horse colours. Molecular genetic studies on coat colour in horses helped in identification of the genes and mutation responsible for coat colour variants. In future, this will be applied to breeding programmes to reduce the incidence of diseases and to increase the efficiency of race horse population.     

Allele frequencies of the extension locus encoding the melanocortin-1 receptor in Japanese and Korean cattle

In order to estimate the influence of the Extension (E) locus in cattle coat color, the melanocortin‐1 receptor (MC1R) gene in Japanese Black, Japanese Brown and Korean (Hanwoo) cattle were sequenced. The sequences of the coding region revealed three alleles (E^D, E⁺ and e), which were previously reported. Polymerase chain reaction‐restriction fragment length polymorphism was performed to investigate the gene frequencies of the three breeds. Japanese Black was almost composed of E^D and E⁺ individuals, E^D = 0.481 and E⁺ = 0.514, and no homozygous e/e, therefore that is consistent with the hypothesis that E^D and E⁺ induce black pigment synthesis. Allele frequencies between Japanese Brown and Hanwoo were obviously different; however, recessive red e allele frequency was 0.038 for Japanese Brown and 0.948 for Hanwoo, even though both breeds have quite similar coat colors (ranging from yellowish brown to dark brown including a red coat color). This result suggested that other genes are also associated with a coat color of red and brown in cattle.     

The relationship of plumage colours with MC1R (Melanocortin 1 Receptor) and ASIP (Agouti Signaling Protein) in Japanese quail (Coturnix coturnix japonica)

1. The relationship of polymorphisms in the Melanocortin 1 Receptor (MC1R) and Agouti Signalling Protein (ASIP) genes with plumage colour in Japanese quail was investigated by cloning and sequencing the entire coding regions from black, white and maroon Japanese quail embryos at 12 d of incubation.

2. Three SNPs were identified in the MC1R coding region by multiple alignment of sequences from individuals with different plumage colours. A missense C/T mutation located at 169 bp within the Open Reading Frame caused a Ile57Val mutation in the amino acid sequence, and had a significant relationship with the black colour.

3. The expression of MC1R was higher in black plumage quails than that in maroon plumage quails, whereas the expression of ASIP was higher in maroon plumage quails than that in black plumage quails.

4. It is concluded that the black plumage colour in Japanese quails may be caused by either increased production of MC1R or decreased production of ASIP.     

阿勒泰白头牛基因组结构变异分析

阿勒泰白头牛是新疆阿勒泰地区的乳肉役兼用地方品种,具有白头、抗病力强和性情温顺特点。结构变异(structure variations, SVs)是影响牛表型性状的重要遗传变异形式。本研究旨在获得阿勒泰白头牛基因组SVs的特征和种质特征的候选基因。以20头阿勒泰白头牛测序深度为17.32X的基因组序列为基础,通过Delly、Lumpy和Manta三个软件进行SVs的检测,并通过SURVIVOR软件取交集进行整合,总共鉴定到37847个SVs。从类型看,缺失、易位、倒位、复制和插入的比例分别为55.5%、26.7%、8.7%、8.9%和不足0.1%。从长度看,缺失、倒位和复制的分布呈现右偏态。以频率大于0.9为标准,获得636个高频SVs,涉及865个蛋白编码基因。基因注释和富集分析发现,阿勒泰白头牛的高频SVs与免疫抗病和神经功能相关,契合了抗病力强和性情温顺特征,可作为相关特征的候选基因(免疫抗病：BolA-DQB、TNIP3、IL1RAP和IL1R1,神经性情：EPHA6、GRM7和HTR2A)。值得注意的是,我们发现ASIP基因5′非编码区存在一个缺失,该基因可减少真黑素的合成,...     

德保矮马与哈萨克马 KIT 基因多态性分析

 马毛色是品种鉴定和个体识别的重要依据。马 KIT 基因位于3号染色体,KIT 基因突变影响马毛色及毛色的分布。对德保矮马和哈萨克马69个个体的 KIT 基因21个外显子及部分内含子直接测序,共发现了5个SNPs,其中1个位于5'-UTR区（g.91214T>G）,1个位于内含子20 （g.171356C>G）,另外3个分别位于外显子15、20和21(g.164297C>T;g.170189C>T;g.171471G>A,p.Ala960Thr)。用PCR-RFLP方法对69个个体进行分型,发现外显子15有3种基因型TT、CT、CC;外显子20有3种基因型TT、CT、CC;外显子21有2种基因型GG与GA,且均为野生型占优势。德保矮马 KIT 基因多态性比哈萨克马更丰富。     

Genetic variation in monoamine oxidase A and analysis of association with behaviour traits in beef cattle

Husbandry of beef cattle requires animals that do not behave aggressively or timidly. The enzyme monoamine oxidase A and the coding gene (MAOA) play an important role in the complex regulation of behaviour. The complete coding region and a part of the non‐coding sequence of the bovine MAOA gene have been analysed in 20 German Angus and 20 German Simmental bulls and cows with the aim of detecting genetic variability. These two cattle breeds are known to differ regarding their behaviour during handling. Five single nucleotide polymorphisms (SNPs) were identified, three of which were found in the coding region of the gene (exons III and XV). One of the SNPs located in exon XV ( NC_007331.3 :g.80340C>T) was found to be a non‐synonymous mutation. The minor allele frequency of this resulting amino acid substitution was significantly different between 543 German Angus and 417 German Simmental calves (0.39 and 0.49, respectively). The potential functional impact of this polymorphism has been tested by in silico analysis, as well as by association analysis using behaviour scores of the genotyped calves for three behaviour tests that assessed the animals’ temperament during tethering, weighing or social separation. In silico analysis did not deliver consistent results arguing for or against a functional impact of the studied amino acid substitution on the function of the biological protein. No significant association was found between this MAOA polymorphism and the behaviour‐related scores analysed in the study.     

CYP19 (cytochrome P450 aromatase) gene polymorphism in murrah buffalo heifers of different fertility performance

The most common cause of infertility in buffaloes is anestrum. During late maturity the ovaries are in a state of true anestrum. One of the predominant causes of true anestrum is a low level of ovarian estrogens. The key enzyme in estrogen biosynthesis is cytochrome P450 aromatase, encoded by CYP19 gene. In the present study, CYP19 gene polymorphism was analyzed by Single Strand Conformational Polymorphism (SSCP) in buffaloes of different fertility performance. The SSCP and sequence analysis revealed 4 allelic variants in coding exons and introns which unaltered the protein sequence. However, a significant polymorphism (T/C heterozygote) was found near TATA binding protein region in regulatory part (a facet of promoter II) at position 23 of CYP19 exon 2, in all late matured and 50% of late maturing animals. Based on these observations and remarks of earlier workers, a hypothesis is proposed for the physiology of late maturity in buffaloes.     

哺乳动物毛色色素Agouti基因位点的研究进展

哺乳动物毛色的形成是由毛囊黑色素细胞产生的真黑素和棕黑素之间的转换而引起的 ,控制哺乳动物毛色的基因位点很多 ,且以错综复杂的方式互相作用。鼠灰色 (Agouti)基因是其中之一 ,结构复杂 ,其位点不是一个单一的基因位点 ,而是一个含有许多等位基因的位点。Agouti基因的表达会引起棕黑素的产生 ,而Agouti不表达时则会引起真黑素的表达 ,从而调节色素合成的真黑素和棕黑素之间的转换。Agouti在鼠皮肤内表达 ,通过对抗黑色素细胞内的黑色素细胞刺激激素受体 (MC1 R)信号来调节毛色色素。Agouti基因位点会发生许多突变 ,一些突变不仅影响毛色 ,而且干扰许多不同的生物学过程。本文就 Agouti基因位点的研究进展进行了综述     

Genetic variation in the 5′UTR of the KRT2.13 gene of sheep

KRT2.13 is a type II keratin wool intermediate filament (IF) protein. Extensive variation was revealed in the 5′ untranslated region (UTR) of the ovine KRT2.13 gene (KRT2.13) using polymerase chain reaction – single strand conformational polymorphism (PCR‐SSCP) analysis. Nine unique PCR‐SSCP patterns were obtained with individual sheep having either one (homozygous), or a combination of two (heterozygous) of these patterns. Seven of the amplicons that produced the apparently homozygous patterns were successfully sequenced (GenBank FJ217670 – FJ217676), revealing eight single nucleotide insertions, 10 single nucleotide substitutions, a nucleotide deletion and a 16 nucleotide insertion that occurred in only one of the sequences. The seven sequences showed between 85% and 95% homology to the previously identified KRT2.13 sequence (GenBank X72379). This study emphasizes the power of PCR‐SSCP analysis in genotyping, as this extensive variation was found in only 100 sheep, of a variety of breeds. Since variation in the 5′UTR of genes may affect their expression, this genetic variation needs to be further studied to establish its role if any, in influencing gene expression and consequently wool traits.     

Coat colour genes in diversity studies

In this paper we describe the use of polymorphic genes affecting coat colour as a tool in diversity studies of domestic animals. Although phenotypic data has been the main criteria for establishing different breeds, calculation of genetic distances between breeds is normally performed using noncoding microsatellite markers. As anticipated, MC1‐R (melanocyte stimulating hormone receptor) allele frequencies vary greatly between cattle breeds expressing different coat colours. In multicoloured breeds, like Icelandic cattle, a high frequency of the E⁺ allele appears to be essential for colour variation. Whereas black breeds have a high frequency of the dominant acting allele E^D, entirely red breeds have no E^D. Animals being homozygous for the defective allele e occurred frequently in some cattle breeds, indicating that the MC1‐R does not have crucial impact on animal physiology other than coat colour. The E⁺ and e alleles were observed in the closely related river buffalo as well. None of the breeds included in this study express the roan phenotype. Consequently, they were monomorphic at the MGF locus. As for the MC1‐R locus, a correlation to colour pattern was observed for two c‐kit alleles as well, confirming that selection of specific phenotypes strongly affect the allelic variation of underlying loci. Information on genes affecting the phenotype is therefore well suited for describing different breeds of livestock and, consequently, a practical tool in breed conservation.     

SSCP技术用于牛mtDNA多态性检测的可行性研究

采用SSCP方法对523头牛线粒体DNA的D-loop序列和12S rRNA基因序列进行多态性研究，再结合DNA直接测序技术发现D-loop序列的SSCP结果与直接测序结果不相符，而12S rRNA的SSCP检测结果与测序结果相同，结果发现对于高变异率片段如线粒体DNA D-loop片段，不适合用 SSCP方法进行多态分析；而对于比较保守的片段，如12S rRNA片段适合用SSCP进行多态性分析。